Method for producing glucose dehydrogenase from Aspergillus oryzae

ABSTRACT

The present invention effectively produces glucose dehydrogenase derived from  Aspergillus oryzae , and provides more practical glucose dehydrogenase.

This application claims priority to U.S. Provisional Applications60/788,252, filed Mar. 31, 2006, and 60/868,276, filed Dec. 1, 2006.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 21,409 bytes ASCII (Text) file named“701383SequenceListing.txt,” created Mar. 28, 2007.

TECHNICAL FIELD

The present invention relates to a gene encoding glucose dehydrogenasederived from Aspergillus oryzae and a method for producing the glucosedehydrogenase by gene recombination.

BACKGROUND ART

Self-monitoring of blood glucose is important for a patient withdiabetes to figure out a usual blood glucose level in the patient andapply it to treatment. An enzyme taking glucose as a substrate isutilized for a sensor used for the self-monitoring of blood glucose. Anexample of such an enzyme includes, for example, glucose oxidase (EC.1.1.3.4). Glucose oxidase is advantageous in that it has highspecificity for glucose and is excellent in thermal stability, and thushas been used as the enzyme for a blood glucose sensor from a long timeago. Its first publication goes back 40 years ago. In the blood glucosesensor using glucose oxidase, the measurement is performed bytransferring electrons produced in a process of oxidizing glucose toconvert into D-glucono-δ-lactone to an electrode via a mediator.However, glucose oxidase easily transfers protons produced in thereaction to oxygen, and thus dissolved oxygen affects the measuredvalue, which has been problematic.

In order to avoid such a problem, for example, NAD(P)-dependent glucosedehydrogenase (EC. 1.1.1.47) or pyrrolo-quinoline quinone-dependentglucose dehydrogenase (EC. 1.1.5.2; former EC. 1.1.99.17) is used as theenzyme for the blood glucose sensor. They dominates in that they are notaffected by dissolved oxygen, but the former NAD(P)-dependent glucosedehydrogenase has the poor stability and requires the addition of thecoenzyme. Meanwhile, the latter pyrrolo-quinoline quinone-dependentglucose dehydrogenase is inferior in substrate specificity, reacts withother sugars such as maltose and lactose and thus correctness of themeasured value is impaired.

In Non-patent documents 1 to 4, glucose dehydrogenase derived fromAspergillus oryzae has been reported, but no glucose dehydrogenase genehas been reported. In Non-patent documents 1 to 4, it has not beendescribed to produce the glucose dehydrogenase derived from Aspergillusoryzae by gene recombination.

-   Non-patent literature 1: Biochim. Biophys. Acta., 1967 Jul. 11;    139(2):265-76-   Non-patent literature 2: Biochim. Biophys. Acta., 1967 Jul. 11;    139(2):277-93-   Non-patent literature 3: Biochim Biophys Acta.146(2):317-27-   Non-patent literature 4: Biochim Biophys Acta.146(2):328-35

In Patent document 1, flavin-binding type glucose dehydrogenase derivedfrom genus Aspergillus has been disclosed. This enzyme dominates in thatthis is excellent in substrate specificity and is not affected by thedissolved oxygen. For the thermal stability, it has been described thata residual activity ratio after being treated at 50° C. for 15 minutesis about 89% and this enzyme is excellent in thermal stability(hereinafter also described as heat resistance). In Patent document 2, agene sequence and an amino acid sequence of that enzyme have beenreported.

-   Patent document 1: WO2004/058958-   Patent document 2: WO2006/101239

Recently, a complete genome sequence of Aspergillus oryzae has beendetermined. However, there is no available information what part of thesequence encodes glucose dehydrogenase.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method forefficiently producing the practically more advantageous enzyme for theblood glucose sensor. More specifically, it is the object to establishthe method for stably producing glucose dehydrogenase derived fromAspergillus oryzae on a large scale by specifying, acquiring andutilizing a gene encoding the glucose dehydrogenase.

For accomplishing the above objects, the present inventors presumed andacquired a glucose dehydrogenase gene derived from Aspergillus oryzae byutilizing database of National Center for Biotechnology Information(NCBI) and found that glucose dehydrogenase derived from Aspergillusoryzae could be acquired from Escherichia coli using the gene.

According to the present invention, by utilizing the glucosedehydrogenase gene isolated from Aspergillus oryzae, it becomes possibleto efficiently produce glucose dehydrogenase and acquire more practicalglucose dehydrogenase.

Thus, the present invention comprises the following

[1] A gene composed of the following DNA (a), (b), (c) or (d):

-   (a) DNA composed of a base sequence described in SEQ ID NO:5;-   (b) DNA composed of a base sequence described in SEQ ID NO:8,    comprising a base sequence described in SEQ ID NO:5 and intron;-   (c) DNA which hybridizes with DNA composed of a base sequence    complementary to DNA (a) under a stringent condition and encodes a    protein having a glucose dehydrogenase activity; or-   (d) DNA which hybridizes with DNA composed of a base sequence    complementary to DNA (b) under the stringent condition and comprises    a region encoding the protein having the glucose dehydrogenase    activity.

[2] A gene encoding the following protein (a) or (b):

-   (a) a protein composed of an amino acid sequence described in SEQ ID    NO:4; or-   (b) a protein composed of an amino acid sequence having one or more    amino acid deletions, substitutions or additions (insertions) in the    amino acid sequence described in SEQ ID NO:4, and having a glucose    dehydrogenase activity.

[3] A recombinant vector comprising the gene according to any of [1] or[2].

[4] A transformant transformed with the recombinant vector according to[3].

[5] The transformant according to [4] wherein a host is Escherichiacoli.

[6] A method for producing a protein having a glucose dehydrogenaseactivity, characterized in that the transformant according to [4] or [5]is cultured in a nutrient medium and the protein having the glucosedehydrogenase activity is collected.

According to the present invention, it has become possible toefficiently produce glucose dehydrogenase. It has become easy to performmolecular biological improvement in order to obtain more practicalglucose dehydrogenase.

BEST MODES FOR CARRYING OUT THE INVENTION

In order to accomplish the above objects, the present inventors found agene DNA presumed to encode glucose dehydrogenase (hereinafter sometimesabbreviated as ATGDH) by utilizing the NCBI database.

A DNA (gene) composed of the base sequence described in SEQ ID NO:1 is agenomic gene sequence comprising a DNA (gene) encoding glucosedehydrogenase derived from Aspergillus oryzae RIB40 strain, predictedfrom the NCBI database, where no intron has been eliminated.

A DNA composed of the base sequence described in SEQ ID NO:2 is obtainedby removing the intron from the sequence of SEQ ID NO:1.

The gene encoding a protein composed of an amino acid sequence describedin SEQ ID NO:3 indicates the entire sequence of a glucose dehydrogenasegene predicted from the NCBI database.

The present inventors predicted from Non-patent literatures 1 to 4 andthe NCBI database that the DNA (gene) encoding the protein having theglucose dehydrogenase activity derived from Aspergillus oryzae could beeasily identified.

And further, they thought that it was also easy that a recombinantvector containing the gene was made, a transformant was made and aprotein encoded by the gene expressed by the transformant was purified.

Although the amino acid sequence and the base sequence encodingflavin-binding glucose dehydrogenase derived from Aspergillus oryzaewere not specified in the NCBI database, specifically, with reference tothe methods described in Non-patent literatures 1 to 4 and publiclyknown technologies, the present inventors tried to culture Aspergillusoryzae, purify glucose dehydrogenase (GDH) from its culture supernatantusing various chromatography methods, make a probe by analyzing itsterminal amino acid sequence and isolate the gene encoding the glucosedehydrogenase.

Likewise, the present inventors tried to isolate the gene encodingglucose dehydrogenase by independently obtaining a microorganismbelonging to genus Aspergillus terreus.

Although the present inventors studied variously, it was found that itwas difficult to obtain a GDH preparation with high purity whose bandcould be clearly identified on SDS-PAGE from the culture supernatant ofAspergillus oryzae TI strain by ordinary purification methods usingsalting out and chromatography methods. It was speculated that sugarchains supposed to be bound to the enzyme protein was one of causes tomake the purification and identification difficult. Therefore, theydetermined that they had no choice but to give up the cloning utilizingthe partial amino acid sequence, which was one of standard methods foracquiring the gene.

Thus, with many trials and errors, it was extremely difficult to acquirethe gene, but as a result of an extensive study, the present inventorsisolated the gene encoding the flavin-binding glucose dehydrogenasederived from Aspergillus oryzae and completed the present invention.

Its detail will be described later in Examples 1 to 3.

One embodiment of the present invention is a method for producing aprotein having a glucose dehydrogenase activity characterized byculturing a transformant transformed with a recombinant vectorcomprising a gene composed of any DNA of the following (a), (b), (c) and(d) or a gene encoding a protein of the following (e) or (f) in anutrient medium and collecting the protein having the glucosedehydrogenase activity.

(a) DNA (gene) composed of a base sequence described in SEQ ID NO:5indicates the entire sequence of DNA (gene) encoding the protein havingthe glucose dehydrogenase activity derived from Aspergillus oryzae TIstrain described later, identified by the present inventors.

(c) DNA (gene) which hybridizes DNA composed of the base sequencecomplementary to DNA composed of the base sequence described in SEQ IDNO:5 under the stringent condition and encodes the protein having theglucose dehydrogenase activity is also included in the presentinvention.

(b) DNA (gene) composed of the base sequence described in SEQ ID NO:8 isa genomic gene sequence comprising DNA (gene) encoding the proteinhaving the glucose dehydrogenase activity derived from Aspergillusoryzae TI strain described later, where no intron has been eliminated.

(d) DNA which hybridizes DNA composed of the base sequence complementaryto DNA composed of the base sequence described in SEQ ID NO:8 under thestringent condition and comprises a region encoding the glucosedehydrogenase activity is also included in the present invention.

(e) The gene encoding the protein composed of the amino acid sequencedescribed in SEQ ID NO:4 indicates the entire sequence of DNA (gene)encoding the protein having the glucose dehydrogenase activity derivedfrom Aspergillus oryzae TI strain described later.

(f) DNA (gene) encoding a protein composed of the amino acid sequencehaving one or more amino acid deletions, substitutions or additions(insertions) in the amino acid sequence described in SEQ ID NO:4 andhaving the glucose dehydrogenase activity is also included in thepresent invention.

Those skilled in the art can easily select the stringent condition bychanging the temperature in the hybridization reaction and the washingand salt concentrations in a hybridization reaction solution and awashing solution. Specifically, the condition where the hybridization isperformed in 6×SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6×SSPE (3MNaCl, 0.2 M NaH₂PO₄, 20 mM EDTA 2Na, pH 7.4) at 42° C. and further thewashing is performed with 0.5×SSC at 42° C. is included as one exampleof the stringent condition of the present invention, but the stringentcondition is not limited thereto. Preferably, the condition where thehybridization is performed in 50% formamide, 6×SSC (0.9 M NaCl, 0.09 Mtrisodium citrate) or 6×SSPE (3 M NaCl, 0.2 M NaH₂PO₄, 20 mM EDTA 2Na,pH 7.4) at 42° C. and further the washing is performed with 0.1×SSC at42° C. is included.

The DNA (gene) of the present invention can include those in which codonusage has been changed to enhance the expression of the GDH.

For example, the above GDH gene derived from Aspergillus oryzae isinserted into an expression vector (many vectors such as plasmids areknown in the art), and an appropriate host (many hosts such asEscherichia coli are known in the art) is transformed with theexpression vector. A water soluble fraction containing GDH can beyielded by culturing the resulting transformant, collecting microbialcells from the medium by centrifugation, disrupting the microbial cellsby a mechanical method or an enzymatic method, e.g., using lysozyme andif necessary adding a chelating agent such as EDTA and a surfactant tosolubilize. Alternatively, by the use of an appropriate host-vectorsystem, it is possible to secret the expressed GDH directly in themedium.

A GDH containing solution obtained as the above could be precipitated byconcentration under reduced pressure, membrane concentration, saltingout treatment using ammonium sulfate or sodium sulfate or fractionalprecipitation using a hydrophilic organic solvent such as methanol,ethanol or acetone. The treatment with heat and isoelectric focusingtreatment are also effective purification procedures. The purified GDHcan also be yielded by performing gel filtration using an absorbingagent or a gel filtration agent, absorption chromatography, ion exchangechromatography and affinity chromatography. It is preferable that thepurified enzyme preparation is purified to an extent that the enzyme isdetected as a single band on electrophoresis (SDS-PAGE).

These can be carried forward in accordance with the followingreferences.

(a) Tanpakushitsu Jikken Protocol Vol. 1, Functional Analysis Vol. 2,Structural Analysis (Shujunsha) edited by Yoshifumi Nishimura and ShigeoOhno.

(b) Revised Tanpakushitsu Jikken Note, Extraction andSeparation/Purification (Yodosha) edited by Masato Okada and KaoriMiyazaki.

(c) Tanpakushitsu Jikken no Susumekata edited by Masato Okada and KaoriMiyazaki.

Alternatively, the above procedures can also be carried forward bymethods exemplified below.

The produced DNA having genetic information of the protein istransferred into the host microorganism by ligating to the vector.

As the vector, those constructed for gene recombination from a phage ora plasmid capable of independently replicating in the host microorganismare suitable. As the phage, for example, Lambda gt10 and Lambda gt11 isexemplified when Escherichia coli is used as the host microorganism. Asthe plasmid, for example, pBR322, pUC19, pKK223-3 and pBluescript areexemplified when Escherichia coli is used as the host microorganism.Among them, those such as pBluescript carrying the promoter capable ofbeing recognized in Escherichia coli upstream of a cloning site arepreferable.

The appropriate host microorganism is not particularly limited as longas the recombinant vector is stable therein and can independentlyreplicate and a trait of a foreign gene can be expressed. ForEscherichia coli, Escherichia coli W3110, Escherichia coli C600,Escherichia coli HB101, Escherichia coli JW109 and Escherichia coli DH5αcan be used.

As the method for transferring the recombinant vector into the hostmicroorganism, for example, when the host microorganism belongs to genusEscherichia, the method of transferring recombinant DNA in the presenceof Ca ions can be employed, and further, an electroporation method maybe used. Furthermore, commercially available competent cells (e.g.,Competent High DH5α supplied from Toyobo Co., Ltd.) may be used. Whenthe yeast is used as the host, a lithium method or an electroporationmethod is used. When filamentous fungus is used, a protoplast method isused.

In the present invention, the method for yielding the gene encoding GDHincludes the following methods. The predicted GDH gene can be found byusing the information for a genomic sequence of Aspergillus oryzae.Then, mRNA is prepared from the microbial cells of Aspergillus oryzaeand cDNA is synthesized. The GLD gene is amplified by PCR using the cDNAobtained in this way as the template, and the recombinant vector isconstructed by binding and closing this gene and the vector at bluntends or sticky ends of both DNA with DNA ligase. The recombinant vectoris transferred into the host microorganism in which the vector canreplicate, and subsequently, the recombinant microorganism containingthe gene encoding GDH is obtained by utilizing a marker of the vector.

GDH in a large amount can be stably produced by culturing themicroorganism which is the transformant yielded as the above in thenutrient medium. The transformant could be selected by searching themicroorganism which has expressed the marker of the vector and the GDHactivity simultaneously. For example, the microorganism which grows in aselection medium based on a drug resistant marker and generates GDHcould be selected.

The base sequence of the GDH gene was decoded by a dideoxy methoddescribed in Science 214:1205, 1981. The amino acid sequence of GDH wasdeduced from the base sequence determined as the above.

As in the above, the once selected GDH gene in the recombinant vectorcan be easily transferred into another recombinant vector which canreplicate in another microorganism by collecting the DNA which is theGDH gene from the recombinant vector carrying the GDH gene byrestriction enzymes and PCR method and binding the DNA to another vectorfragment. For the transformation of another microorganism with thesevectors, the competent cell method by treating with calcium, theelectroporation method and the protoplast method can be used.

The GDH gene of the present invention may be those having the DNAsequence so that a part of amino acid residues is deleted or substitutedin the amino acid sequence after translation of the gene or so thatother amino acid residues are added or substituted, as long as theprotein encoded by the GDH gene has the glucose dehydrogenase activity.

As the method for modifying the gene encoding the wild type GDH, thetypically performed technique to modify the genetic information is used.That is, DNA having the genetic information of the modified protein ismade by converting the specific base in DNA having the geneticinformation of the protein or inserting or deleting the specific base.The specific methods for converting the base in the DNA include the useof commercially available kits (Transformer Mutagenesis Kit suppliedfrom Clonetech; EXQIII/Mung Bean Deletion Kit supplied from Stratagene;QuickChange Site Directed Mutagenesis Kit supplied from Stratagene), orutilization of polymerase chain reaction (PCR) method.

For the culture of the host microorganism which is the transformant, aculture condition could be selected in consideration of nutritionalphysiological natures of the host. It is advantageous that thetransformant is cultured in liquid culture in many cases andindustrially ventilation stirring culture is performed. But, consideringthe productivity, it is more advantageous in some cases that thefilamentous fungus is used as the host and a solid culture is employed.

As nutrient sources of the medium, those typically used for the cultureof the microorganism can be widely used. Carbon sources may be carboncompounds capable of being assimilated. For example, glucose, sucrose,lactose, maltose, lactose, molasses and pyruvic acid are used. Nitrogensources may be usable nitrogen compounds. For example, peptone, meatextracts, yeast extracts, casein hydrolyzed products, and bean cakeextracted with alkali are used. In addition, phosphate salts, carbonatesalts, sulfate salts, salts of magnesium, calcium, potassium, iron,manganese and zinc, particular amino acids and particular vitamins areused if necessary.

A culture temperature can be appropriately changed in the range in whichthe microorganism grows and produces GDH, and is preferably about 20 to37° C. A culture time period is somewhat different depending on thecondition, the culture could be terminated at an appropriate time periodby judging the time to be right to reach the maximum yield of GDH, andthe culture time period is typically about 6 to 48 hours.

A pH value in the medium can be appropriately changed in the range inwhich the microorganism grows and produces the GDH, and is preferably inthe range of about pH 6.0 to 9.0.

The culture medium containing the microbial cells which produce GDH canbe directly collected and utilized. However, in general, according tostandard methods, when the GDH is present in the culture medium, aGDH-containing solution is separated from the microorganism microbialcells by filtration or centrifugation, and subsequently utilized. WhenGDH is present in the microbial cells, the microbial cells are collectedfrom the culture by filtration or centrifugation, then disrupted by themechanical method or the enzymatic method using lysozyme and ifnecessary the chelating agent such as EDTA and the surfactant are addedto solubilize, and GDH is separated/collected as an aqueous solution.

The GDH-containing solution obtained as the above could be precipitatedby concentration under reduced pressure, membrane concentration, saltingout treatment using ammonium sulfate or sodium sulfate or fractionalprecipitation using the hydrophilic organic solvent such as methanol,ethanol or acetone. The treatment with heat and isoelectric focusingtreatment are also effective purification procedures. The purified GDHcan also be yielded by subsequently performing gel filtration using theabsorbing agent or the gel filtration agent, absorption chromatography,ion exchange chromatography and affinity chromatography.

For example, it is possible to obtain a purified enzyme preparation byseparating and purifying by gel filtration using Sephadex gel (suppliedfrom GE Health Care Bioscience), or column chromatography using DEAESepharose CL-6B (supplied from GE Health Care Bioscience) or OctylSepharose CL-6B (supplied from GE Health Care Bioscience). It ispreferable that the purified enzyme preparation is purified to theextent that the enzyme is detected as a single band on electrophoresis(SDS-PAGE).

In the present invention, the glucose dehydrogenase activity is measuredunder the following condition.

<Reagents>

-   50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)-   14 mM 2,6-dichlorophenol-indophenol (DCPIP) solution-   1 M D-glucose solution.-   A reaction reagent is made by mixing 15.8 mL of the PIPES buffer,    0.2 mL of the DCPIP solution and 4 mL of the D-glucose solution.    <Measurement Condition>

The reaction reagent (2.9 mL) is preliminarily heated at 37° C. for 5minutes. The GDH solution (0.1 mL) is added and gently mixed,subsequently the change of absorbance at 600 nm is recorded for 5minutes using a spectrophotometer controlled to 37° C. using water as acontrol, and the change of absorbance per one minute (ΔOD_(TEST)) iscalculated from a linear portion of the record. The solvent in which GDHwill be dissolved in place of the blinded GDH solution is added to thereagent mixture, and the change of absorbance (ΔOD_(BLANK)) per oneminute is measured. The GDH activity is calculated from these valuesaccording to the following formula. One unit (U) in the GDH activity isdefined as the amount of the enzyme which reduces 1 μM DCPIP for oneminute in the presence of 200 mM D-glucose.Activity (U/mL)=[−(ΔOD_(TEST)−ΔOD_(BLANK))×3.0×dilutionscale]/(16.3×0.1×1.0)

In the above formula, 3.0 represents a liquid amount (mL) of thereaction reagent+the enzyme solution, 16.3 represents a millimolarmolecular absorbance coefficient (cm²/μmol) in the condition ofmeasuring the present activity, 0.1 represents the liquid amount of theenzyme solution (mL) and 1.0 represents a light path length (cm) of thecell.

EXAMPLES

The present invention will be more specifically described below byExamples, but the present invention is not limited to the followingExamples.

An outline of the procedure to acquire the GDH gene derived fromAspergillus oryzae described in Examples shown below is as follows.

In order to acquire the GDH gene derived from Aspergillus oryzae, thepurification of GDH from the culture supernatant of Aspergillus oryzaeand Aspergillus terreus was tried using salting out, chromatography andthe like, but it was difficult to yield GDH with high purity (Example 1[1])

Therefore, we had no choice but to give up the cloning utilizing thepartial amino acid sequence, which was one of standard methods toacquire the gene.

Thus, we searched GDH-producing microorganisms other than the abovemicroorganisms, and as a result of an extensive study, we found thatPenicillium lilacinoechinulatum NBRC6231 produced GDH, and succeeded toyield the purified enzyme with high purity from the culture medium ofthis fungal strain (Example 1 [2]).

Subsequently, we succeeded to determine the partial amino acid sequenceusing the above enzyme, partially acquired the GDH gene derived from P.lilacinoechinulatum NBRC6231 by PCR based on the determined amino acidsequence and determined its base sequence (1356 bp) (Example 1 [3] and[4]).

Finally, based on this base sequence, the GDH gene derived fromAspergillus oryzae was presumed (Example 1 [5]) from the publisheddatabase of Aspergillus oryzae genome, and it was acquired.

Example 1 Estimation of Glucose Dehydrogenase Gene Derived fromAspergillus oryzae (Hereinafter Sometimes Abbreviated as “AOGDH”)

-   [1] Acquisition of GDH Derived from Aspergillus oryzae

Aspergillus oryzae obtained from soils and stored as dried microbialcells according to standard methods was used. This is referred to asAspergillus oryzae TI strain below.

Aspergillus oryzae TI strain was restored by inoculating its drymicrobial cells in the potato dextrose agar medium (supplied from Difco)and incubating at 25° C. Fungal threads restored on the plate werecollected including the agar, which was then suspended in filtratedsterilized water. In two 10 L jar fermenters 6 L of a production medium(1% malt extract, 1.5% soy bean peptide, 0.1% MgSO₄.7H₂O, 2% glucose, pH6.5) was prepared and sterilized by autoclave at 120° C. for 15 minutes.After cooling, the above fungal thread suspension was inoculated, andcultured with ventilation and stirring at 30° C. The culture was stopped64 hours after the start of the culture, and a filtrate containing theGDH activity was collected by removing the fungal threads by filtration.Low molecular substances were removed from the collected supernatant byultrafiltration (molecular weight 10,000 cut off). Then, ammoniumsulfate was added at 60% saturation to perform ammonium sulfatefractionation. The supernatant containing the GDH activity was collectedby centrifugation, adsorbed to the Octyl-Sepharose column, and elutedwith ammonium sulfate having the gradient from 60% saturation to 0% tocollect fractions having the GDH activity. The resulting GDH solutionwas applied onto the G-25 Sepharose column to perform the salting out.Ammonium sulfate was added at 60% saturation thereto. The mixture wasadsorbed to the Phenyl-Sepharose column and eluted with ammonium sulfatehaving the gradient from 60% saturation to 0% to collect fractionshaving the GDH activity. The fraction having the GDH activity was heatedat 50° C. for 45 minutes, and then centrifuged to yield the supernatant.The solution obtained from the above steps was made a purified GDHpreparation (AOGDH). In the above purification process, 20 mM potassiumphosphate buffer (pH 6.5) was used as the buffer. In order to determinethe partial amino acid sequence of the AOGDH, the further purificationwas tried using various procedures such as ion exchange chromatographyand gel filtration chromatography, but no purified preparation capableof being subjected to the partial amino acid sequencing could beobtained.

Also, we independently searched and obtained the microorganism belongingto Aspergillus terreus, and likewise tried the purification from itsculture supernatant by the salting out and the Octyl-Sepharose, but nopurified preparation capable of being subjected to the partial aminoacid sequencing could be obtained as was the case with Aspergillusoryzae. Typically, using the purification methods commonly used, it ispossible to obtain the protein preparation with high purity detected asa clear single band on SDS-PAGE. However, the GDH preparation at such alevel could not be obtained. It was speculated that one of its causeswas the sugar chain thought to be bound to the enzyme protein.Therefore, we had no choice but to give up the cloning utilizing thepartial amino acid sequence of the protein, which was one of standardmethods to acquire the gene.

-   [2] Acquisition of GDH Derived from Filamentous Fungus Belonging to    Genus Penicillium

A purified preparation detected to be nearly uniform on SDSelectrophoresis was acquired by using Penicillium lilacinoechinulatumNBRC6231 as the GDH producing fungus derived from the filamentous fungusbelonging to genus Penicillium and performing the culture and thepurification according to the same procedure as in the case with theabove Aspergillus oryzae.

-   [3] Preparation of cDNA

For Penicillium lilacinoechinulatum NBRC6231, according to the abovemethods, the culture was carried out (but, the culture in the jarfermenter was performed for 24 hours), and the fungal threads werecollected by filter paper filtration. The collected fungal threads wereimmediately frozen in liquid nitrogen, and disrupted using Cool Mill(supplied from Toyobo Co., Ltd.). The total RNA was immediatelyextracted from disrupted microbial cells using Sepasol RNA I (suppliedfrom Nacalai Tesque) according to the protocol of this kit. mRNA waspurified from the resulting total RNA using Origotex-dt30 (supplied fromDaiichi Pure Chemicals Co., Ltd.), and RT-PCR with this as the templatewas performed using ReverTra-Plus™ supplied from Toyobo Co., Ltd. Aresulting product was electrophoresed on agarose gel and a portioncorresponding to a chain length of 0.5 to 4.0 kb was cut out. cDNA wasextracted from a cut out gel fragment using MagExtractor-PCR&Gel CleanUp supplied from Toyobo Co., Ltd. and purified to use as a cDNA sample.

-   [4] Determination of GDH Gene Partial Sequence

The purified GDH derived from NBRC6231 was dissolved in Tris-HCl buffer(pH 6.8) containing 0.1% SDS and 10% glycerol, and partially digested byadding Glu specific V8 endoprotease at a final concentration of 10 μg/mLthereto and incubating at 37° C. for 16 hours. This sample waselectrophoresed on 16% acrylamide gel to separate peptides. Peptidemolecules present in this gel were transferred on a PVDF membrane usingthe buffer for blotting (1.4% glycine, 0.3% Tris and 20% ethanol) bysemi-dry method. The peptides transferred onto the PVDF membrane werestained using a CBB staining kit (GelCode Blue Stain Reagent suppliedfrom PIERCE), two band portions of the visualized peptide fragments werecut out and internal amino acid sequences were analyzed using a peptidesequencer. The resulting amino acid sequences were IGGWVDTSLKVYGT (SEQID NO:9) and WGGGTKQTVRAGKALGGTST (SEQ ID NO:10). Based on thissequence, degenerate primers containing mixed bases were made, and PCRwas performed using the cDNA derived from NBRC6231 as the template. Anamplified product was obtained, and was detected as a single band ofabout 1.4 kb by agarose gel electrophoresis. This band was cut out, andextracted and purified using MagExtractor-PCR&Gel Clean Up supplied fromToyobo Co., Ltd. The purified DNA fragment was TA-cloned using TArgetClone-Plus, and Escherichia coli JM 109 competent cells (Competent HighJM109 supplied from Toyobo Co., Ltd.) were transformed with theresulting vector by heat shock. Among transformed clones, for coloniesin which an insert had been identified by blue-white determination, theplasmid was extracted and purified using MagExtractor-Plasmid byminiprep, and the base sequence (1356 bp) of the insert was determinedusing plasmid sequence specific primers.

-   [5] Estimation of AOGDH Gene

Based on the determined base sequence, the homology was searched on thehome page of “NCBI BLAST” (http://www.ncbi.nlm.nih.gov/BLAST/), and theAOGDH gene was estimated from multiple candidate sequences inconsideration of the homology to publicly known glucose oxidationenzymes. The homology of the AOGDH estimated from the search to the GDHpartial sequence derived from P. lilacinoechinulatum NBRC6231 was 49% atan amino acid level.

Example 2 Acquisition of AOGDH Gene and Introduction into Escherichiacoli

For obtaining the AOGDH gene, mRNA was prepared from the microbial cellsof Aspergillus oryzae TI strain, and cDNA was synthesized. Two oligo DNArepresented by SEQ ID NOS:6 and 7 were synthesized, and the AOGDH genewas amplified using the prepared cDNA as the template and using KOD PlusDNA polymerase (supplied from Toyobo Co., Ltd.). A recombinant plasmidwas constructed by treating the resulting DNA fragment with Nde I andBam H I and inserting it into Nde I-Bam H I sites in pBluescript (theNde I site had been introduced to match a Nde I recognition sequence ATGto a translation initiation codon ATG of LacZ). Escherichia coli DH5α(supplied from Toyobo Co., Ltd.) was transformed with this recombinantplasmid. The plasmid was extracted from the transformant according tothe standard method, and the base sequence of the AOGDH gene wasdetermined (SEQ ID NO:5). As a result, it was found that the amino acidsequence deduced from the cDNA sequence was composed of 593 amino acidresidues (SEQ ID NO:4). GDH predicted from RIB40 strain registered inthe database is composed of 588 amino acid residues (SEQ ID NO:3),suggesting that the GDH from RIB40 strain and the GDH from TI strain aredifferent in amino acid residue number. For the gene, the sequence wasidentified using TI strain genomic DNA, and the gene flanking regionswere identified using RACE method. It was suggested that RIB40 strainwhich was the database strain and TI strain were different in GDH genesequence. Thus, using the recombinant plasmid containing TI strain GDHgene as the template, the recombinant plasmid containing the GDH genesequence predicted from the sequence of the database RIB40 strain wasmade using QuickChange Site Directed Mutagenesis Kit (supplied fromStratagene), and the transformant was acquired. These transformants werecultured with shaking in a liquid medium (Terrific broth) containing 100μg/mL of ampicillin at 30° C. for 16 hours. When the GDH activity wasmeasured in disrupted microbial cell solutions, no GDH activity could beidentified in the transformant having the GDH sequence derived fromRIB40 strain whereas the high GDH activity of 8.0 Upper mL of theculture medium was obtained in the transformant having the GDH sequencederived from TI strain. The GDH activity in the culture supernatant ofAspergillus oryzae TI strain in Example 1 was 0.2 U/mL. These resultssuggested that the GDH gene predicted from the RIB40 database sequencedid not function as GDH. So far as the gene sequences of TI strain andRIB40 strain were compared, it was thought that the partial deletion inthe GDH gene sequence derived from RIB40 strain caused it.

Example 3

The Escherichia coli DH5α transformant having the GDH sequence derivedfrom TI strain cultured in Example 2 was collected by centrifugation,the microbial cells were suspended in 20 mM potassium phosphate buffer(pH 6.5), and then disrupted using French press to extract GDH. This wastreated by the same procedure as in AOGDH purified in Example 1 to yielda purified enzyme preparation (rAOGDH). Its properties were comparedwith those of the AOGDH purified enzyme preparation.

-   [1] Substrate Specificity

When the blood glucose level is measured using a blood glucose sensor,it is required to use the enzyme specific for glucose not to lead tomisdiagnosis. Thus, the reactivity of rAOGDH to various sugars wasexamined. The results are shown in Table 1. It was confirmed that rAOGDHand AOGDH exhibited the equivalent substrate specificity and did not actupon maltose to which reactivity was particularly problematic when thepatient using transfusion used the sensor.

TABLE 1 Aspergillus oryzae Recombinant AOGDH rAOGDH Glucose 100.0 100.0Maltose 0.4 0 Fructose 0 0.2 Arabinose 0 0.3 Glycerin 0.1 0.2 Sucrose0.1 0 Melezitose 0 0.5 Sorbose 0 0 Ribose 0 0.1 Maltotriose 0.2 0.2Maltotetraose 0.7 0.2 Galactose 0.4 0.6 Mannose 0.8 2.1 Trehalose 0.51.0

-   [2] Maltose Degradability

Even if GDH itself used for the blood glucose sensor does not act uponmaltose, when the component to degrade maltose into glucose is containedin the enzyme preparation, it potentially leads to the misdiagnosis.That is, it is extremely important that the component to degrade maltoseis not contained in the GDH enzyme preparation. Thus, the contaminationof the component to degrade maltose in the GDH enzyme preparation wasexamined. A test for the contamination of the component to degrademaltose was performed as follows. Maltose (8 mM, 50 μL) was added to 50μL of each purified enzyme solution prepared at 10 U/mL, and reacted at37° C. After the completion of the reaction, the concentration ofglucose contained in the reaction solution was examined using Liquitecglucose HK test (supplied from Roche Diagnostics). A standard curve forcalculating the glucose concentration was made by measuring glucosesolutions for setting a standard coefficient (supplied from Wako PureChemical Industries Inc.). The results are shown in Table 2.

TABLE 2 Reaction time Purified enzyme 30 sec 60 sec 3 min 10 min Freeglucose AOGDH 7.10 7.15 7.37 7.89 concentration rAOGDH 0    0.030  0.0050   (mM) (recombinant) Maltose AOGDH 89%  89%  92%  99%  degradationrAOGDH 0%   0.38%  0.06% 0%  ratio (recombinant)

In the AOGDH purified preparation, after treating at 37° C. for 30seconds, 90% of maltose was already degraded into glucose, and afterreacting for 10 minutes, nearly 100% maltose was degraded. Thus, thesame measurement was performed using the preparation obtained by highlypurifying AOGDH for the purpose of the analysis of the partial aminoacid sequence in Example 1, but the activity to degrade maltose wasidentified. Meanwhile, in the rAOGDH preparation, even after treating at37° C. for 10 minutes, the accumulation of glucose was not observed atall. Aspergillus oryzae has been utilized for fermentation industry froma long time ago, and has been known to produce sugar-related enzymessuch as amylase and glucoamylase in large amounts. Thus, it is thoughtthat it is extremely difficult to purify only GDH at high purity undersuch an environment.

From these results, it is thought that it is essential to use GDHprepared from the recombinant gene when GDH derived from Aspergillusoryzae is used for the blood glucose sensor.

INDUSTRIAL APPLICABILITY

The present invention enables to produce glucose dehydrogenase derivedfrom Aspergillus oryzae on a large scale by the use of recombinantEscherichia coli. By the present invention, it becomes possible toproduce glucose dehydrogenase which does not act upon maltose in a broadsense and is suitable for the glucose sensor and the like.

1. An isolated gene composed of the following DNA (a) or (b): (a) DNAcomposed of a base sequence described in SEQ ID NO: 5; or (b) DNAcomposed of a base sequence described in SEQ ID NO:
 8. 2. An isolatedgene encoding a protein composed of an amino acid sequence described inSEQ ID NO:
 4. 3. A recombinant vector comprising the gene according toclaim
 1. 4. A host cell transformed with the recombinant vectoraccording to claim
 3. 5. The host cell according to claim 4, wherein thehost cell is Escherichia coli.
 6. A method for producing a proteinhaving a glucose dehydrogenase activity, comprising culturing the hostcell according to claim 4 in a nutrient medium such that the proteinhaving glucose dehydrogenase activity is produced and collecting theprotein.
 7. A recombinant vector comprising the gene according to claim2.
 8. A host cell transformed with the recombinant vector according toclaim
 7. 9. The host cell according to claim 8, wherein the host cell isEscherichia coli.
 10. A method for producing a protein having a glucosedehydrogenase activity, comprising culturing the host cell according toclaim 5 in a nutrient medium such that the protein having glucosedehydrogenase activity is produced and collecting the protein.
 11. Amethod for producing a protein having a glucose dehydrogenase activity,comprising culturing the host cell according to claim 8 in a nutrientmedium such that the protein having glucose dehydrogenase activity isproduced and collecting the protein.
 12. A method for producing aprotein having a glucose dehydrogenase activity, comprising culturingthe host cell according to claim 9 in a nutrient medium such that theprotein having glucose dehydrogenase activity is produced and collectingthe protein.